Use of inhibitors for the treatment of disorders related to RTK hyperfunction, especially cancer

ABSTRACT

The present invention concerns the use of inhibitors for the treatment and/or prophylaxis of diseases which are the consequence of increased receptor tyrosine kinase activity, particularly cancer. The use is particularly directed towards inhibition or lowering of the overexpression and/or altered activity of receptor tyrosine kinases (RTKs). In particular, this altered activity of receptor tyrosine kinase can be triggered by a mutation of FGFR-4, wherein this mutation is in particular a point mutation in the transmembrane domain of FGFR-4 and leads to an exchange of a hydrophobic amino acid for a hydrophilic amino acid. The invention further concerns the use of an inhibitor directed against FGFR-4, for the treatment and/or prophylaxis of cancer. Furthermore, the invention concerns a mutated FGFR-4, which leads to overexpression and/or altered activity in cells. Finally, the invention concerns a DNA and RNA sequence of a mutated FGFR-4 molecule. Finally, in addition the invention concerns a pharmaceutical composition, containing the inhibitor as described above and further a diagnostic and screening procedure.

The present invention concerns the use of inhibitors for the treatmentand/or prophylaxis of diseases which are the consequence of increasedreceptor tyrosine kinase activity, particularly cancer. The use isparticularly directed towards inhibition or lowering of theoverexpression and/or altered activity of receptor tyrosine kinases(RTKs). In particular, this altered activity of receptor tyrosine kinasecan be triggered by a mutation of FGFR-4, wherein this mutation is inparticular a point mutation in the transmembrane domain of FGFR-4 andleads to the exchange of a hydrophobic amino acid for a hydrophilicamino acid. The invention further concerns the use of the inhibitors ofFGFR kinases, particularly for the treatment and/or prophylaxis ofcancer. Furthermore, the invention concerns a mutated FGFR-4, whichleads to overexpression and/or altered activity in cells. Finally, theinvention concerns a DNA and RNA sequence of a mutated FGFR-4 molecule.Finally, in addition the invention concerns a pharmaceuticalcomposition, containing the inhibitor as described above and, further, adiagnostic and screening procedure.

Cell growth is a carefully regulated process dependent on the specificneeds of an organism. In a young organism, the cell division rateexceeds the cell death rate, which leads to an increase in the size ofthe organism. In an adult organism, the new formation of cells and celldeath are balanced so that a “steady state” arises. In rare cases,however, the control of cell multiplication breaks down and the cellsbegin to grow and to divide, although no specific need for a highernumber of cells of this type exists in the organism. This uncontrolledcell growth is the cause of cancer. Factors that can provoke theuncontrolled cell growth, sometimes associated with metastasisformation, are often of a chemical nature, but can also be of a physicalnature, such as for example radioactive radiation. Another cause of thetriggering of cancer are genetic peculiarities or mutations in a certainorganism, which sooner or later lead to the cells degenerating.

Up to now, it has still not been possible satisfactorily to elucidatethe processes which control normal growth and differentiation, forexample in the breast. In addition to hormonal control, there is also acomplex network of different, locally generated growth factors whichintervene in the development of the mammary cells. The precise causes ofthe occurrence of cancer in mammary cells are as unclear and unknown asthey are diverse, as is also the case with other cells. Alterations inoncogenes and tumour suppressor genes appear to play an important partin breast cancer carcinogenesis. In addition, reinforced stimulation byregulatory factors which arise in genetically altered cells can lead toincreased progression of cell growth

At present, essentially two alternatives are available for the treatmentof cancer. Either the cancer cells are successfully removed from thediseased organism completely by a surgical intervention, or attempts aremade to render the degenerated cells in the organism harmless, forexample by administration of medicaments (chemotherapy) or by physicaltherapeutic procedures, such as irradiation.

In chemotherapy, medicaments are often used which in some form intervenein the DNA metabolism and damage rapidly growing cells, which have toproduce higher DNA metabolic capacity, more strongly than cells whichare dividing slowly or not at all. However, a severe disadvantage ofmany chemotherapeutic drugs is the low specificity of the activesubstance used, as a result of which healthy cells are also damagedduring the chemotherapy. This low specificity of the active substancesfurther requires that their dosage must in each case be such that as fewas possible healthy cells are damaged, with simultaneous killing of thecancer cells. This is often not possible, and the cancer patient diesbecause of the ever further spreading cancer cells, which in the finalstages cause the failure of vital functions.

It is assumed that the overexpression and/or altered activity of certaingrowth factor receptors contribute to the intensified growth of manyneoplasms, including breast cancer. For example, the overexpression ofEGFR, i.e. epidermal factor receptor, or ERB B-2 receptor in breasttumours has been linked with a poor prognosis. FGF (historically:fibroblast growth factor) proteins could also be involved in thedevelopment of cancer in breast glands or of other cancer; however theresults in this regard are contradictory or are inconclusive.

The FGFs constitute a large family of peptide regulatory factors, ofwhich 9 members are so far known. Eight of these have been wellcharacterised in man (Basilico and Moscatelli, 1992; Coulier et al.,1993). The FGFs operate via high-affinity tyrosine kinase receptors,which are coded for by at least four different genes. Further, the FGFsare multifunctional, regulatory peptides which could have an effect notonly on tumorigenesis but could also play a major part in cardiovasculardiseases, reconstruction after tissue injury, neurobiology and embryonicdevelopment. The acidic and basic FGFs (aFGF and bFGF) were the firstand are the best characterised members of the family. In vivo it couldfor example be shown that FGFs are involved in mesodermal induction inembryogenesis (Slack et al., 1987; Kimelman et al., 1988), and alsoinvolvement in angiogenesis (Thomas et al., 1985; Thompson et al., 1989;Folkmann and Klagsbrun, 1987).

For the corresponding receptors (FGFRs), four similar genes coding forthem have been identified. These genes code for structurally relatedproteins with an extracellular domain which consists of threeimmunoglobulin loops and an acid portion, a hydrophobic trans-membranedomain and an intracellular domain, which incorporates a tyrosine kinaseactivity. For two of these genes, FGFR-1 and FGFR-2, it could be shownthat they have multiple transcripts, which arise by alternative splicing(Givol and Yayon, 1992 and Johnson and Williams, 1993). Splice variantswhich arise from these genes differ with respect to the number ofinmmunoglobulin-like domains in the extracellular region of the receptorand in the sequence for the second half of the third immunoglobulindomain, which can arise from alternative exons. In addition,transmembrane and juxtamembrane shortenings or deletions can arise,which can generate secreted or kinase-inactive protein products.

For FGFR-3, it was possible to find alternative transcripts andcorresponding isoforms, but for FGFR-4 there is only a single knownprotein product. Because of the large number of FGFR genes andtranscripts and the lack in many protein products of a specificity fordefined FGFs, it is difficult to determine the action of a specificligand on a specific receptor. Hence, correlations between specific FGFreceptors and defined diseases can only be established with greatdifficulty, let alone a correlation of a particular mechanism of actionof a defined receptor with a disease. Accordingly, it is difficulteffectively to treat diseases, especially the complex disease picturecancer, utilising the FGFRs.

Hence it is an objection of the present invention to specify a possibletreatment and/or prophyl-axis of somatic disorders, in the developmentof which receptor tyrosine kinases (RTKs) are involved, particularlycancer. In particular, it is an objection of the present invention toinhibit and/or to lower overexpression and/or altered, for exampleconstitutive activity of receptor tyrosine kinases.

It is further an objection of the present invention to inhibit and/or tolower the altered activity of the receptor tyrosine kinase of a mutatedFGFR-4.

It is a further an objection of the present invention to specify afurther RTK which is involved in carcinogenesis and/or metastasisformation. Further, it is an objection of the present invention tospecify a DNA sequence or corresponding RNA sequence of the RTK.

It is a further an objection of the present invention to specifyimproved diagnostic or differential diagnostic and screening procedures.

Finally it is an objection of the present invention to specify apharmaceutical composition, with which in particular cancer can betreated.

These objections are achieved by the objects of the independent claims.The dependent claims specify preferred developments of the invention.

For the better understanding of the present invention, the terms usedherein are explained in more detail.

By “inhibitor” is understood any substance which inhibits the RTK orlowers their activity. This can be a low-molecular weight substancedirected against the RTK, a kinase-inactive receptor or an anti-receptorantibody.

By “kinase inactive receptor” is understood any receptor which no longerhas any tyrosine kinase activity.

By “receptor tyrosine kinase” [sic] is understood any receptor which hastyrosine kinase activity. The expression includes growth factorreceptors which have tyrosine kinase activity, and also HER2 or themet-receptors.

By “RTK-Hyperfunction” is understood overexpression (see below) and/oraltered activity (see below).

“Defective signal transfer activities” means that a mutated receptor isno longer capable of converting an extracellular growth signal oranother signal into an intracellular signal, in the sense that thisdefective signal generation no longer depends on the presence of aligand, for example the growth factor.

“Growth factor” means any mitogenic chemical, usually a polypeptide,which inter alia is secreted by normal and/or transformed mammaliancells, and which plays a significant part in the regulation of cellgrowth, in particular in the stimulation of the proliferation of thecells and the maintenance of their viability. The term “growth factor”for example includes epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF) and nerve growth factor (NGF), and also FGF, namelyfibroblast growth factor.

By “mutated receptor tyrosine kinase” is understood a receptor tyrosinekinase which by comparison with the wild type receptor contains astructural alteration, so that the receptor has a different, e.g. nolonger regulable, tyrosine kinase activity from the wild type receptor.One class of mutations leads to altered activity of the RTK.

By “wild type growth factor receptor” or “wild type” receptor isunderstood a naturally occurring growth factor receptor or receptor thatbears the non-mutated amino acid sequence. The “wild type” correspondsto the receptor variant most commonly occurring in the population.

By “extracellular domain” of the growth factor receptor or receptor isunderstood the part of the receptor which normally projects out of thecell into the extracellular surroundings. The extracellular domain forexample includes the part of the receptor to which a growth factor oranother molecule (ligand) binds.

By “transmembrane region” of the growth factor receptor or receptor isunderstood the hydrophobic portion of the receptor, which is normallylocated in the cell membrane of the cell which expresses the receptor.

By “tyrosine kinase domain” or “cytoplasmic domain” of the growth factorreceptor or receptor is understood the portion of the receptor which isnormally situated inside the cell, and brings about thetransphosphorylation of tyrosine residues.

By “an effective quantity” is understood a quantity of the compositionaccording to the invention which can achieve the desired therapeuticeffect.

By “fibroblast growth factor (FGF)” is understood a mitogenicpolypeptide which influences the growth and other properties of cells,inter alia of fibroblasts.

By “overexpression” is understood increased production of RTK protein bya cell as compared to the wild type. This can for example be triggeredby gene amplification of the RTK gene and lead to excessive,uncontrolled cell division activity.

By “altered activity” is understood permanent activity of a signaltransfer route mediated by growth factor receptors. Thus with an alteredRTK the kinase activity is also present when no ligand is present.

According to the present invention, it could be shown that a mutatedFGFR-4 can lead to overexpression and/or altered activity of thecorresponding receptor tyrosine kinase in cells and hence lead tocancer.

Growth factor receptors play a decisive part in the development andmultiplication of human cancer cells. In healthy cells, the growthfactor receptors are inter alia involved in the control of cell growth,but also in differentiation, cell migration, etc. The actual signal forthe cell division is the growth factor, which is formed depending on theneeds of the organism. The receptor undertakes the function of signaltransfer, i.e. it is involved in the conversion of the extracellulargrowth signal into cell division activity in the inside of the cell.With many growth factor receptors, their ability, after binding of thegrowth factor to the extracellular domain, to transfer phosphateresidues onto tyrosine residues in proteins plays a decisive part. Thesereceptors are also described as receptor tyrosine kinases. A review ofreceptor tyrosine kinases is to be found in Yarden Y and Ullrich A, Rev.Biochem. 1988, 57, 443-78. The dimerisation of these growth factorreceptors after binding of the growth factor is a further importantevent in the process of signal transfer. The conversion of anextracellular signal into an intracellular signal mediated by growthfactor receptors with tyrosine kinase activity can be broken down intothe following five steps:

1. The binding of the growth factor (also described as ligand) to theextracellular domain of the receptor induces a conformational change;this causes

2. dimerisation of receptors with altered conformation; with

3. simultaneous induction of kinase activity;

4. transphosphorylation of tyrosine residues in the receptor dimer,which once again creates and stabilises an activated receptorconformation; and

5. phosphorylation of polypeptide substrates and interaction withcellular factors.

Uncontrolled hyperfunction of this signal transfer chain for examplebecause of the overexpression or altered activity of the receptor caninter alia lead to increased division activity of the relevant cells andin the extreme case to a degenerated cancer cell. A review concern-inggrowth factor receptors and their function in signal transfer from theextracellular to the intracellular milieu, and the possible influence ofabnormally expressed receptors on carcino-genesis, is given in Ullrich Aand Schlessinger J (1990) Cell 61, 203-212.

It has now surprisingly been found that in the five-stage signaltransfer chain explained above, a mutated FGFR-4 results in increasedsignal transfer activity, in the development of which the alteredactivity of mutated RTK is decisively involved.

Hence according to claim 1 of the present invention at least oneinhibitor of a receptor tyrosine kinase is used for the treatment and/orprophylaxis of RTK-hyperfunction-induced disorders, particularly cancer.Furthermore, according to the invention diseases or somatic disorderswhich are the consequence of a hyperproliferation of tissues and/orincreased invasivity of tissues attributable to increased signaltransfer can also be eliminated or alleviated.

As inhibitor, as well as low-molecular weight substances, for example atleast one kinase-inactive receptor can be used. Through the use of theinhibitor, e.g. of the kinase-inactive receptor, the altered activity ofthe receptor tyrosine kinase can be inhibited and/or lowered. As hasalready previously been stated, the overexpression and/or alteredactivity of growth factor receptors is an important factor in thetriggering or the progression of cancer. The overexpression of EGFR orthe Erb B-2 receptor in breast tumours has for example been associatedwith a poor prognosis (see above). Hence inhibition of thisoverexpression and/or altered activity is an important component in thetreatment and/or prophylaxis of cancer. FGFR-4 is tissue-specificallyswitched off during embryogenesis. However, it is present in 30% ofbreast cancer patients; it is not detectable in the tissue of healthysubjects. The use of inhibitors for receptor tyrosine kinase leads to alowering or complete inhibition of the overexpression and/or alteredactivity. Likewise, the use of kinase-inactive receptors leads to alowering and/or complete inhibition of the activity of the receptortyrosine kinases, since the kinase function of the heterodimer is nolonger capable of signal transfer. The action of kinase-inactivereceptors is based on the fact that non-functional heterodimers areformed (dilution effect). A lack of signal transfer leads to preventionof the transmission of the overexpressed and/or altered active signal,as a result of which the signal is prevented from conversion into abiological response of the cell. As a result, through this inhibition ofthe receptor tyrosine kinase or through these kinase-inactive receptors,it is possible effectively and positively to intervene in the treatmentand/or prophylaxis of cancer.

It has surprisingly been found that the FGFR-4 mutation also occurs inthe germ line of healthy persons. It is assumed that the germ linemutation leads to a genetic predisposition, which renders the personsconcerned susceptible to the outbreak of various diseases. In connectionwith carcinogenesis, it is assumed that the increase expression of themutated receptor in the tumour tissue is involved in the carcinogenesis.The germ line mutation is further regarded as a predisposition interalia for the following diseases: arteriosclerosis, leukaemia, lymphoma,hepatic cell carcinoma and cholangiocarcinoma.

Consequently, the present invention makes a further genetic markeravailable, which is found to be extremely helpful in the diagnosis andearly recognition of various diseases and susceptibility to these.

The present invention therefore also concerns a procedure for thedetection of a nucleic acid which codes for FGFR-4 in case material,whereby in particular mutations of the receptor-coding nucleic acid aredetected. This can for example be effected by hybridisation witholigonucleotide probes, which can specifically indicate the presence orabsence of a mutation, in particular a point mutation. In this, forexample a “mismatch” between mutated nucleic acid and oligonucleotide isutilised such that if a “mismatch” is present a hybridisation does nottake place and hence there is no signal. Alternatively, mutations canalso be detected by amplification of the nucleic acid with specificFGFR-4 PCR primers and subsequent cleavage with suitable restrictionendonucleases. If for example a mutation affects the recognitionsequence of a restriction endonuclease, such that for example themutated recognition sequence is no longer recognised as a cleavage siteby the restriction endonuclease, this leads to a different restrictionfragment than in the non-mutated wild type. By means of the PCR,restriction fragments can be specifically detected, so that in thestated case for example a larger restriction fragment is present in themutant compared to the wild type. Alternatively, however, a mutation canalso lead to the creation of a new restriction cleavage site, as aresult of which a “wild-type fragment” after cleavage with theappropriate enzyme becomes smaller in the mutant. The mutation in thetransmembrane domain of FGFR-4, at position 388 of the sequence, asdeposited in the EMBL Gene Bank/DDBJ under X57205, which leads to anexchange of Gly in the wild type for Arg in the mutant (SEQ ID NO: 1),concerns the recognition sequence GGWCC of the restriction endonucleaseBstN1. As a result, two new restriction fragments of 80 and 29 b.p. areformed, which can inter alia be detected by restriction analysis.

According to the present invention, it could further be shown thatoverexpression and in particular altered activity of the RTK leads toincreased invasivity, i.e. to intensified metastasis formation. Sincemetastasis formation is one of the main problems of cancer, this meansthat the inhibition or lowering of the overexpression and/or alteredactivity will lead to an effective agent in the combating of cancer, bywhich in particular metastasis formation is inhibited.

Possible inhibitors are for example described in Mohammadi et al.(1997).

Preferably according to the invention an intervention is made into anoverexpression and/or altered activity of the receptor tyrosine kinase,which is triggered by a mutation of FGFR-4. This mutation can be one orseveral point mutations. In particular, the mutation/mutations occur inthe transmembrane domain of FGFR-4, as a result of which in particular ahydrophobic amino acid is exchanged for a hydrophilic amino acid.

It is already known that point mutations which have led to an exchangeof hydrophobic for hydrophilic amino acids in FGFR-3 are associated withcertain diseases. Thus for example, an altered activity of fibroblastgrowth factor receptor 3 due to a point mutation in the transmembranedomain has been found in achondroplasia. Achondroplasia, which is themost commonly occurring genetic form of dwarfism, is an autosomaldominant disorder, which is essentially based on a defect in thematuration process of certain bones. It could be shown thatachondroplasia is triggered by a Gly to Arg substitution in thetransmembrane domain of FGFR-3. It could further be shown that that theArg mutation in FGFR-3 activates the kinase function of the dimericreceptor. The Arg point mutation also leads to a ligand-independentstimulation of the tyrosine kinase activity of FGFR-3 itself and tostrongly increased altered levels of phosphotyrosine on the receptor.These results suggest that the molecular basis of achondroplasia isunregulated signal transfer by FGFR-3.

A further mutation in the transmembrane domain of FGFR-3 is an exchangealanine for glutamine. This amino acid exchange leads to anotherdisease, namely to Crouzon's disease with acanthosis nigricans.

According to the present invention, it was established that mutations inFGFR-4, especially point mutations in the transmembrane domain, whichlead to an exchange of a hydrophobic for a hydrophilic amino acid, areinvolved in the triggering and poor prognosis for cancer, on account ofwhich inhibition of receptor tyrosine kinases or the use ofkinase-inactive receptors are suitable for the treatment and/orprophylaxis of cancer, wherein the receptor tyrosine kinases areoverexpressed or active in an altered way owing to a mutation.

In particular, for the point mutation at position 388, which leads to anexchange of glycine for arginine, it could be shown that as a result ofthis the receptor tyrosine kinases become active in an altered way, andthis homo- and heterozygotically results in signal transfer withoutligand stimulation, as a result of which in turn an uncontrollablegrowth of cells can be triggered. In the worst case, this uncontrolledgrowth leads to cancer. The transmembrane domain then has the sequence(ID No.1):

RYTDIILYASGSLALAVLLLLARLY,

while the non-mutated domain has the following sequence (ID No.2):

RYTDIILYASGSLALAVLLLLAGLY.

Without being bound to one theory, it is assumed that the activation ofthe receptor tyrosine kinase which bears one of the aforesaid pointmutations and in particular the point mutation at position 388, whichleads to an exchange of glycine for arginine, is based on astabilisation of the receptor in a dimeric conformation, which occursbecause of interactions through which changes in the transmembranedomain were made possible. The intensified formation of aligand-independent dimer leads to increased receptor tyrosine kinaseactivity and cellular transformation. Other possibilities for the effectof the point mutations on the triggering of cancer may for example havea basis in that the mutation acts on the signal transfer by FGFR-4, inthat the receptor migration through the membrane is prevented, thereceptor dimerisation with itself or with other FGFRs is disturbed, orin that the tyrosine kinase activity of the receptor is affected.

According to the present invention, it could be shown that 56% ofpatients from St Petersburg with breast tumours (study of biopsies)carried the mutation at position 388, which is linked with an exchangeof glycine for arginine. Of these, 45% were heterozygotic and 11%homozygotic. This significantly high proportion suggests a link betweenthe point mutation at position 388 and the occurrence of breast cancer.

In a further study, in which German patients with breast tumours werestudied, only 43% of the patient showed the point mutations at position388. From the study with normal tissues of cancer patients and DNA fromthe tissue of normal individuals, it can be inferred that the mutationis a germ line mutation.

Furthermore, genomic DNA and cDNA from cell lines was also studied, inorder to determine the proportion of point mutations at position 388.The cell lines studied derived from breast tumours, normal breastepithelial cell lines as a comparison, squamous cell carcinoma,glioblastomas, neuroblastomas and uterine cancer. With all cell lines,except for the normal breast epithelial cell lines, a significantpercentage of the point mutation at position 388 in the FGFR-4 moleculecould be found. Hence the above-mentioned use of inhibitors orkinase-inactive receptors is especially suitable for the treatment ofcarcinomas. Here the treatment of neuroblastomas, uterine cancer andpancreatic cancer, but also other types of cancer, seems especiallypromising.

Particularly preferred is the use of inhibitors which inhibit a mutatedFGFR-4, especially with the mutation Gly→Arg at position 388 in thetransmembrane domain.

Further, the present invention concerns a mutated FGFR-4, which leads tooverexpression and/or altered activity of the receptor in cells.Preferably, this mutated FGFR-4 is characterised in that a hydrophobicamino acid in the wild type receptor has been exchanged for ahydrophilic amino acid in the mutated receptor. Especially preferred isa mutation which is a point mutation and occurs in the transmembranedomain. Still more preferably, the point mutation occurs at position388, as a result of which preferably a glycine is replaced by arginine.

Hitherto, it was assumed among experts that only one FGFR-4 occurs,which is not mutated. Mutated FGFR-4 was unknown. It was thereforesurprising that it could be shown according to the present inventionthat a mutated FGFR-4 exists. In particular, according to the presentinvention a connection between the mutations, in particular the pointmutation at position 388, and the occurrence of cancer could bedemonstrated. Furthermore, the germ line mutation in healthy persons hasbeen connected with the genetic predisposition for the occurrence interalia of arteriosclerosis.

The invention further concerns a DNA molecule containing a sequencewhich codes for a mutated FGFR-4. The invention also includes an RNAmolecule, containing an RNA sequence which codes for a mutated FGFR-4.The above sequences can be used for diagnosis of cancer. In this, thesequences can specifically recognise the mutations in the FGFR-4. Thepresence of the mutation in the FGFR-4 is linked with a poor prognosisfor the treatment of the cancer. The reason for this could be aggressivegrowth behaviour of the corresponding tumour.

Apart from this, the invention concerns a procedure for the differentialdiagnosis of breast cancer, wherein the patient's nucleic acid isbrought into contact with one of the DNAs and/or RNAs described above,so that a signal is obtained, which indicates the presence and/orabsence of mutated FGFR-4. Finally, the present invention concerns apharmaceutical composition, containing the inhibitor or thekinase-inactive receptor, as described above. Apart from this, theinvention concerns a screening procedure for the identification ofinhibitors of tyrosine kinase activity, wherein the receptor accordingto the invention is brought into contact with potential inhibitors andthe tyrosine kinase activity in the presence and/or absence of theinhibitor is determined.

Further, the detection of the presence of a mutation can also beperformed by PCR and subsequent restriction enzyme cleavage, as alreadydescribed in more detail above.

Finally, other molecular biological diagnostic procedures are also apossibility.

Further, the object of the invention is an antibody which specificallyreacts with a mutated FGFR-4 according to the invention. “Specific” inthe sense of the invention means that the antibody according to theinvention binds to the mutated, but not to the non-mutated, receptor.

Below, the invention is described in detail by the figures and examples.

Here:

FIG. 1 shows SDS PAGE of an immunoprecipitation of FGFR-4 (thephosphorylated FGF receptor-4 is marked by an arrow),

FIG. 2 a polyacrylamide gel of the mutated FGFR-4,

FIG. 3 a sequence analysis of the transmembrane domain of FGFR-4,

FIG. 4 the correlation between the FGFR-4 mutation G388R and the lymphnode metastasis formation status (n=number of patients, p=P value) and

FIG. 5 the correlation between the FGFR-4 mutation G388R and therelapse-free survival time (n=number of patients, p=P value).

EXAMPLES

Cell Culture. The human cell lines MDA-MB-453, ZR 75-1, K562 and SKBr3were obtained from the ATCC. The individual supply sources can be foundin the table at the end. MDA-MBA453, K562 and ZR 75-1 were cultivated inRPMI (Gibco, Eggenstein) containing 10% foetal calf serum (Sigma,Taufkirchen). SKBR3 was cultivated in McCoy's 5a (Gibco, Egenstein)containing 15% foetal calf serum. All cell culture media containedpenicillin streptomycin (Sigma, Taufidrchen). The cells were incubatedat 37° C. in a water-vapour saturated atmosphere and 8% CO₂.

Cloning of FGFR-4^(388Arg)/wt. For preparation of RNA from K562 andMDA-MB453 cells, 3×10⁷ cells were lysed with guanidinium isothiocyanateand purified by ultracentrifugation in a CsCl gradient. The cDNAsynthesis was effected with reverse transcriptase (Boehringer, Mannheim)and 10 pmol of “random oligonucleotides” in each case, according to themanufacturer's instructions. 0.5 μl were used in a subsequent PCRreaction.

FGFR-4³⁸⁸Arg and FGFR-4wt were amplified by the PCR reaction. For this,the following primers were used: sense-GCTCAGAGGGCGGGCGGGGGTGCCGGCCG[SEQ ID NO: 3]; anti-sense CCGCTCGAGTGCCTGCACAGCCTTGAGCCTTGC [SEQ ID NO:4]. For the PCR reaction, the following were used: 1.5 U/25 μlExpand-Polyrnerase (Boehringer, Mannheim) and reaction buffer accordingto the manufacturer's instructions: 200 μM dNTP's; 0.01% v/v TritonX100; 10% v/v DMSO, and 0.2 pmol each of sense and α-sense primer. Thefollowing reaction steps were performed: 35 cycles, 94° C. 1 min, 64° C.1 min, 72° C. 2.5 min. MDA-MB-453 cDNA was used for the cloning ofFGFR-4^(388Arg), and K562 cDNA for the cloning of FGFR-4 wt. The PCRproducts were cloned in the pcDNA3 vector (Invitrogen). In this way,both a FGFR-4 with the G388R and also a wild type FGFR-4 could beobtained for further tests.

Amplification of the transmembrane domain of FGFR-4. The followingprimers were used: sense-GACCGCAGCAGCGCCCGAGGCCAG [SEQ ID NO: 5];anti-sense AGAGGGAAGAGGGAGAGCTTCTG [SEQ ID NO: 6]. For the PCR reaction,the following were used: 1.5 U/25 μl , Taq-Polymerase (Boehringer,Mannheim) and reaction buffer according to the manufacturer'sinstructions: 200 μM dNTP's; 0.2 pmol each of sense and α-sense primer,0.5 μl cDNA or genomic DNA from tumour biopsies and cell lines; thefollowing reaction steps were performed: 35 cycles, 95° C. 45 secs, 72°C. 45 secs.

Analysis by restriction digestion. The transmembrane domain of FGFR-4from genomic or cDNA was amplified as described above. To test biopsiesand cell lines for the G1217A mutation by restriction digestion, the PCRproducts were incubated for 1 hr at 60° C. with 5 U/25 μl of BstN1 (NEB,Schwalbach/Taunus). The DNA fragments from the restriction digestionwere separated with a 20% polyacrylamide gel and stained with ethidiumbromide. The analysis of the wild type receptor yields a 109, a 37 and a22 base-pair sized fragment (track 4). On the other hand, as a result ofthe mutation G1217A a further restriction cleavage site for BstN1 isformed. The mutated receptor shows further 80 and 29 base-pair sizedfragments, while the 109 base-pair sized fragment disappears (track 1:homozygotic; tracks 2 and 3: heterozygotic) (see FIG. 2).

Genotype analysis of genomic DNA by restriction digestion. Genomic DNAfrom the tissue samples of the primary turnouts was isolated by standardmethods (Current Protocols in Molecular Biology, John Wiley and Sons,Inc., 1995). In order to be able to genotype analyse the genomic DNA,the transmembrane region in the FGFR-4 gene was amplified with thefollowing primers in a PCR reaction: 5′-GACCGCAGCAGCGCCCGAGGCCAG-3′ (bp1129-1142; [SEQ ID NO: 5]), and 5′-AGAGGGAAGAGGGAGAGCTTCTG-3′ (bp1275-1297; [SEQ ID NO: 6]). For the PCR reaction, Ready-to-Go PCR Beats(Pharmacia, Uppsala, Sweden) were used. The following PCR cycles wereused: 3 min at 95° C., 45 secs at 94° C., 45 secs at 72° C. and 5 minsat 72° C. A total of 35 cycles were performed. The PCR products wereincubated for I hr at 60° C. with 5 U/25 μl of BstN1 (NEB,Schwalbach/Taunus). The DNA fragments from the restriction digestionwere separated with a 20% polyacrylamide gel and stained with ethidiumbromide. The ³⁸⁸Arg allele is characterized by two fragments of 80 and29 bp size, while the ³⁸⁸Arg allele is indicated by a single 109 bpsized fragment. Each genotype analysis was repeated three times.

DNA sequencing of PCR products. For the sequence analysis of thetransmembrane domain of FGFR-4, the PCR products were cloned into theBluescript vector. For this, a PCR reaction was performed as alreadydescribed. The following primers were used:sense-GGGAATTCGACCGCAGCAGCGCCCGAGG [SEQ ID NO: 7];α-sense-GCTCTAGAAGAGGGAAGAGGGAGAG [SEQ ID NO: 8]. The PCR products ofthe cloning of FGFR-4^(Arg388)/wt could be directly sequenced in thevector pcDNA3. The DNA sequencing of plasmid DNA was performed by thechain termination method. After annealing of the T/-primer onto theplasmid DNA, the sequencing reaction was performed with T/-DNApolymerase (Pharmacia, Freiburg). The products of the sequencingreaction were then separated on a denaturing 5% polyacrylamide gel (7.5M urea; 1×TBE) and exposed on Xray film after drying (see FIG. 3). Fromthis, the DNA sequences of the wild type and also of the mutation, wereobtained.

Immunoprecipitation and Western blot analysis. 2.2×10⁶ cells were spreadonto 10 cm Petri dishes and incubated overnight. Then the cell mediumwas replaced by medium with no foetal bovine serum and incubated for afurther 24 hrs. For the stimulation, the cells were incubated for 10mins with 50 ng aFGF/ml, washed twice with cold PBS and placed on ice.The cells were incubated for 15 mins at 4° C. with 300 μl of cold lysisbuffer (1% w/w NP-40, 1.25% w/v sodium deoxycholate, 0.1% w/v SDS, 0.15M NaCl, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 10 mM sodiumfluoride, 1 mM PMSF, 20 μg/ml aprotinin, 1 mM orthovanadate, 10 mMsodium pyrophosphate), and the lysate clarified by centrifugation(13,000 RPM) at 4° C. For the protein value determination, the Micro-BCAProtein Assay (Pierce) was used in accordance with the manufacturer'sinstructions. For the immuno-precipitation, the cell lysates wereadjusted to equal protein content and then incubated for 18 hrs at 4° C.with 0.5 μg anti-FGFR-4 (Santa Cruz) and protein-A-Sepharose (PharmaciaFreiburg) on a rotating wheel. The immune complexes were washed 4 timeswith cold HNTG (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton X100, 10%glycerine, 10 mM sodium pyrophosphate). For sample preparation, theimmune complexes were treated with 50 μl 3×Laemmli buffer and incubatedfor 5 mins at 99° C. The precipitated proteins were separated on a 7.5%SDS-PAGE (see FIG. 1).

For Western blots, the proteins separated by SDS-PAGE were transferredto nitrocellulose. Non-specific protein binding sites on the membranewere blocked by incubation for 2 hrs at room temperature withTBS-T/0.25% gelatine (10 mM Tris/HCl pH 8.0, 0.15 M NaCl, 0.05%Tween20). The incubation with primary antibodies was effected for 6 hrsat 4° C. on a tilt shaker. Non-specifically bound antibodies were thenremoved by washing 4 times with TBS-T/0.25% gelatine. The binding ofsecondary antibodies was effected for 1 hr at room temperature. Thenon-specifically bound secondary antibodies were removed by a furtherwashing step. Immune complexes were made visible with the ECL™ kit(Amersham, Braunschweig) in accordance with the manufacturer'sinstructions.

Statistical Methods. Statistical calculations were performed with theaid of the statistics program MedCalc (MedCalc Software, Belgium) andEpiInfo 6.04b (CDC, Atlanta, Ga.). In order to determine thecorrelations between the genotypes in the different patient groups andthe clinical data, an odds ratio, the confidence interval (CI) and astatistical significance (P value) were calculated. Because of the smallnumber of ³⁸⁸Arg homozygotic patients, this group was combined with thegroup of ³⁸⁸Arg heterozygotic patients for the statistical calculations.

Detection of FGFR-4 in Tumour Cel Lines. Table 1 shows the correlationbetween the expression of RTK and breast cancer. Expression of RTKclearly occurs more often in cell lines from breast cancer, while noexpression is detectable in cell lines of normal breast epithelialcells.

TABLE 1 Detection of FGFR-4 in Breast Cancer Cells Northern Blot [sic]Breast Cancer Cell Lines FGFR-4 1 HTB-30 (SK-BR-3) ++ 2 HTB-122 (BT-549)− 3 MCF-7 + 4 BT-483 +++ 5 T-47D + 6 ZR-75-1 +++ 7 MDA-MB-468 − 8MDA-MB-453 ++++ 9 MDA-MB-361 ++++ 10 MDA-MB-415  − 11 MDA-MB-231  −Normal Breast Epithelial Cell Lines 12 HBL-100  − 13 MCF-10A  − Key: −:no expression +: expression ++: strong expression +++: very strongexpression ++++: extreme expression.

From Table 2, it is clear that the G388R mutation also occurs in celllines of other cancer types and is correlatable with these. In healthyepithelial cell lines, the mutation is not detectable.

TABLE 2 Mutation FGFR-4 G388R in various other tumour cell lines Samplegenomic DNA cDNA Glioblastoma U-138 −/− −/− U-373 −/− −/− U-172 −/− −/−U-118 −/* −/* SF-763 −/− −/− U-1240 */* */* T-98G (*)/− (*)/− U-937 −/−−/− Neuroblastoma SK-N-SH −/* −/* SH-SY-SY −/* −/* Uterine Cancer OAW-42−/* −/* PA-1 −/− −/− Caov-3 −/− −/− Squamous Hlac-78 −/− −/− Hlac-79 −/−−/− Scc-4 −/− −/− Scc-10a −/− −/− Scc-10b −/− −/− Scc-17a −/− −/−Scc-17b −/− −/− Scc-22a */* */* Scc-22b */* */* HaCat −/− −/− FaDu −/−−/− Normal Breast Epithelial Cell Lines HBL-100 −/− −/− MCF-10A −/− −/−Key: −/− homozygotically nonmutated */− heterozygotically mutated */*homozygotically mutated

Detection of the FGFR-4 Mutation G388R in Biopsies. Table 3 thus showsthat of 61 female patients from St Petersburg with breast cancer whowere studied, 56% carried the G388R mutation, 45% of themheterozygotically and 11% homozygotically. Of the 69 female breastcancer patients from Munich who were studied, 43% carried the G388Rmutation, 32% of them heterozygotically and 11% homozygotically. Theproportion of the total percentage of the mutation in female patientsfrom St Petersburg and Munich is different. This suggests that the G388Rmutation is a germ line mutation.

TABLE 3 Detection of FGFR-4 mutation G388R in biopsies Samples frombreast tumours From St Petersburg From Munich Sample gen. DNA Samplegen. DNA cDNA 19 102 T */− 5382 T */− */− 20 102 N 5609 T */* */* 21 103T */− 8926 T */− */− 22 103 N */− 9456 T */* */* 23 2 T */− 9556 T */−*/− 24 2 N */* 10347 T −/− −/− 25 12 T −/− 10555 T −/− −/− 26 12 N 10681T */− */− 27 13 T −/− 10781 T */− */− 28 13 N 10808 T */* */* 29 14 T*/− 11189 T */− */− 30 14 N */− 11526 T */− */− 31 15 T −/− 11697 T */−32 15 N 11820 T −/− −/− 33 17 T */− 12015 T −/− −/− 34 17 N */− 12166 T*/− */− 35 18 T −/− 13932 T */− */− 36 18 N 16003 T */− */− 37 20 T −/−16353 T −/− −/− 38 20 N 1 N 39 21 T */* 2 T */− */− 40 21 N */* 3 N 4122 T */− 4 T −/− −/− 42 22 N 5 N 43 23 T */− 6 T −/− −/− 44 23 N 7 N 4531 T */− 8 T −/− −/− 46 31 N */− 9 N 47 42 T −/− 10 T */− */− 48 42 N 11N 49 43 T −/− 12 T −/− −/− 50 43 N 13 N 51 45 T −/− 14 T */− */− 52 45 N16 T */− */− 53 47 T */− 17 N −/− 54 47 N */− 18 T */− 55 48 T −/− 19 N56 48 N 20 T −/− 57 50 T −/− 38 T −/− 58 50 N 3433 T −/− 59 53 T −/−3539 T 60 53 N 3631 T */* 61 54 T */− 3632 T −/− 62 54 N */− 3636 T −/−63 55 T −/− 3637 T */− 64 55 N 3638 T −/− 65 60 T */− 3640 T −/− 66 60 N*/− 991 N −/− 67 61 T */* 991 T −/− 68 61 N */* 15153 N 69 62 T */−15153 T −/− 70 62 N */− 15856 N */* 71 63 T */− 15856 T */* 72 63 N */−12845 N 73 67 T */− 12845 T −/− 74 67 N */− 19044/93 N 75 69 T −/−19044/93 T −/− 76 69 N 9426/93 N 77 78 T */− 9426/93 T −/− 78 78 N */−2005 N */− 79 79 T */* 2005 T */− 80 79 N */* 14860 N 81 82 T −/− 14860T −/− 82 82 N 4198 T −/− 83 83 T −/− 5739 T */* 84 83 N 6060/93 tum */*85 85 T −/− 6982/93 tum −/− 86 85 N 7244/93 tum −/− 87 86 T */− 8114/93tum −/− 88 86 N */− 8335/93 tum */− 89 87 T −/− 8481/93 tum */− 90 87 N8566/93 tum −/− 91 89 T −/− 8786/93 tum */* 92 89 N 9145/93 tum −/− 9394 T −/− 9354/93 tum −/− 94 94 N 9796/93 tum −/− 95 97 T */* 9798/93 tum−/− 96 97 N */− 10125/93 tum −/− 97 98 T −/− 10150/93 tum */− 98 98 N11218/93 tum −/− 99 99 T */* 11673/93 tum −/− 100 99 N */* 13232/93 tum−/− 101 100 T −/− 13316/93 tum */− 102 100 N 14724/93 tum −/− 103 101 T*/− 14879/93 tum #1 −/− 104 101 N */− 14879/93 tum #2 −/− 105 102 T(#20) */− 15645/93 tum −/− 106 102 N (#19) */* 107 103 T (#22) */− 108103 N (#21) 109 104 T */− 110 92 T */* 111 65 T 112 52 T */− 113 35 T*/− 114 33 “A” T */− 115 33 “B” mts. */− 116 30 T */− 134 30 N */− 11727 T */* 133 27 N */* 118 24 T */− 119 10 T */− 132 10 T */− 120 3 T −/−121 90 T −/− 122 90 N 123 80 T −/− 124 80 N 125 81 T −/− 126 58 T −/−127 51 T */− 128 51 N 129 44 T */− 130 44 N

Correlation between the FGFR-4-G388R mutation and the detection ofFGFR-4 expression. From Table 4 below, it is clear that the G388Rmutation (genomic DNA and cDNA) only occurs when expression and/orintensified expression occurs. The mutation is detectable neither in thenormal breast epithelial cell lines nor in the breast cancer cell linesin which no RTK expression was found.

The cell line MDA-MB 453, whose RTK expression is especially pronounced,shows a homozygotic G388R mutation.

TABLE 4 Correlation between the FGFR-4-G388R mutation and the detectionof FGFR-4 expression Northern Blot Mutation Breast cancer cell lineFGFR-4 cDNA gen. DNA 1 HTB-30 (SK-BR-3) ++ +/+ +/+ 2 HTB-122 (BT-549) −−/− −/− 3 MCF-7 + +/− +/− 4 BT-483 +++ +/− +/− 5 T-47D + +/− +/− 6ZR-75-1 +++ +/− +/− 7 MDA-MB-468 − −/− −/− 8 MDA-MB-453 ++++ +/+ +/+ 9MDA-MB-361 ++++ +/− +/− 10 MDA-MB-415 − −/− −/− 11 MDA-MB-231 − +/− +/−Normal Breast Epithelial Cell Lines 12 HBL-100 − −/− −/− 13 MCF-10A −−/− −/− Key: −: no expression +: expression ++: strong expression +++:very strong expression ++++: extreme expression. −/−: no mutation */−:heterozygotically mutated */*: homozygotically mutated

Study of the Correlation Between the Occurrence of the FGFR-4 MutationG388R and Lymph Node Metastasis Status or Relapse-Free Survival Time

Table 5 shows the clinical parameters of all patients who took part inthe study of the role of the G388R mutation in the tumorigenesis ofbreast cancer. It is found that patients with a G388R mutation have aworse long-term prognosis than patients with no G388R mutation.

Key to Table 5: [on following pages]

Her2: expression level of the Her2 receptor; 0=no expression to3=overexpression

OPDAT: date of the operation

M/R: metastasis formation/relapse; 0=no/1=yes

Vers: died; 0=no/1=yes

ÜBERRE: survival time without relapse, in months

Grade: differentiation grade of tumour; 1=strong differentiation/3=lowdifferentiation

Stage: size of the primary tumour.

E-Rec: expression of the oestrogen receptor, 0=no expression to12=highest expression

GEN: genotype of the FGFR-4; G=wild type allele; R=mutated allele

BEDAT: date of last observation

REZDAT: date of relapse diagnosis

TODDAT: date of death

ÜBERLEB: survival time overall

Nod.: metastases in the lymph nodes; 0=no/1=yes

Men: menopause

P-Rec: expression of the progesterone receptor: 0=no expression to12=highest expression

TABLE 5 FGFR-4 genotypes and clinical case data Patho Nr. Her2 GEN OPDATBEDAT M/R REZDAT Vers TODDAT ÜBERRE 489292 G/G 31.03.92 14.02.97 0 0 59617792 G/G 24.04.92 14.07.94 0 0 27 639792 0 G/G 29.04.92 03.04.94 0 023 724493 G/G 11.05.93 23.06.97 0 0 48 914593 0 G/G 16.06.93 07.03.96 00 33 935493 2+ G/G 21.06.93 15.03.96 0 0 33 963392 G/G 30.06.92 30.06.970 0 60 979693 G/G 29.06.93 07.03.96 0 0 33 979893 1+ G/G 29.05.9330.06.97 0 0 48 1034792 0 G/G 13.07.92 26.03.97 0 0 56 1323293 G/G02.03.93 05.08.97 0 0 48 1331693 3+ G/G 30.09.92 29.03.95 0 0 36 15645931+ G/G 19.10.92 19.06.97 0 0 56 78692 G/G 19.01.88 29.06.93 0 0 65176989 3+ G/G 08.02.85 06.02.92 0 0 84 273690 1+ G/G 22.02.86 14.02.92 00 72 725289 G/G 14.12.87 0 729991 0 G/G 23.05.87 22.02.92 0 0 57 7332901+ G/G 28.05.86 28.03.87 0 1 28.03.87 14 826790 2+ G/G 19.06.86 22.01.940 0 91 867191 0 G/G 19.06.87 22.01.94 0 0 79 988590 G/G 24.07.8622.01.94 0 0 90 991790 0 G/G 23.07.86 25.03.92 0 0 68 1031190 2+ G/G30.07.86 26.01.94 0 0 90 1033391 1+ G/G 21.07.87 03.03.92 0 0 55 1055592G/G 14.07.88 25.04.93 0 1 25.04.93 57 1101191 0 G/G 31.07.87 03.06.93 00 70 1426491 1+ G/G 08.10.87 04.02.94 0 0 76 1560492 G/G 22.09.8802.04.92 0 0 42 1605790 0 G/G 25.11.86 20.02.92 0 0 63 1641488 2+ G/G21.12.84 04.02.94 0 0 109 1121893 1+ G/G 23.07.93 18.01.96 1 06.09.94 118.01.96 14 1201592 1+ G/G 10.08.92 25.04.95 1 25.04.95 0 29 258093 0G/G 22.02.85 18.04.91 1 30.11.89 1 18.04.91 57 479090 3+ G/G 04.04.8623.07.89 1 12.04.88 1 23.07.89 24 806490 0 G/G 14.06.86 18.09.90 110.11.88 1 18.09.90 29 963589 1+ G/G 26.07.85 06.03.89 1 26.04.88 106.03.89 33 972291 3+ G/G 09.07.87 19.08.89 1 16.07.88 1 19.08.89 12995589 0 G/G 01.08.85 27.06.88 1 29.04.87 1 27.06.88 21 1112389 3+ G/G30.08.85 14.07.86 1 14.06.86 1 14.07.86 9 1726892 G/G 18.11.88 23.04.911 07.11.90 1 23.04.91 24 289791 3+ G/R 25.02.87 02.04.92 0 0 61 337293G/R 03.03.89 04.03.92 0 0 36 879290 2+ G/R 01.07.86 01.10.92 0 101.10.92 75 893090 0 G/R 03.07.86 17.12.93 0 0 89 1106192 G/R 23.07.8804.02.94 0 0 66 1107789 G/R 29.08.85 13.02.92 0 0 77 1113892 G/R24.07.92 14.10.97 0 0 63 1118990 G/R 19.08.86 15.02.92 0 0 66 1152692 3+G/R 31.07.92 30.06.97 0 0 59 1599789 1+ G/R 14.12.85 15.02.87 0 115.02.87 14 1614591 0 G/R 12.11.87 25.02.92 0 0 51 92390 0 G/R 20.01.8613.02.92 1 09.02.91 0 61 99289 0 G/R 23.01.85 17.03.92 1 22.02.90 0 61130588 2+ G/R 20.01.84 30.01.92 1 18.02.87 0 37 306490 3+ G/R 01.03.8606.06.87 1 06.11.86 1 06.06.87 8 492191 3+ G/R 07.04.87 20.04.94 110.10.90 0 42 529692 G/R 07.04.92 29.03.96 1 23.02.95 1 29.03.96 34529990 3+ G/R 17.04.86 29.04.87 1 14.04.87 1 29.04.87 12 538292 3+ G/R07.04.92 13.10.93 1 07.04.92 1 13.10.93 0 614091 2+ G/R 29.04.8721.04.90 1 31.07.88 1 21.04.90 15 651591 0 G/R 07.05.87 17.09.93 114.04.90 0 35 673592 G/R 06.05.92 27.02.96 1 06.06.93 0 13 678092 2+ G/R07.05.92 29.06.96 1 07.05.92 0 0 714289 2+ G/R 04.06.85 16.09.89 130.06.87 1 16.09.89 25 807492 0 G/R 29.05.92 29.11.96 1 29.11.96 0 54848193 G/R 03.06.93 10.09.94 1 16.08.94 1 10.09.94 15 955692 1+ G/R29.06.92 26.11.96 1 17.08.95 37 1022090 0 G/R 29.07.86 02.07.88 123.06.88 1 02.07.88 23 1054987 G/R 05.08.83 15.12.84 1 29.02.84 115.12.84 7 1078192 2+ G/R 20.07.92 22.10.95 1 25.09.95 0 38 1079689 2+G/R 22.08.85 27.03.91 1 31.12.89 1 27.03.91 52 1169792 2+ G/R 04.08.9201.07.95 1 01.07.95 1 01.07.95 47 1216692 0 G/R 12.08.92 30.07.96 109.09.94 0 23 1314689 2+ G/R 16.10.85 21.02.92 1 05.11.91 0 73 13919922+ G/R 17.09.92 01.05.93 1 15.01.93 1 01.05.93 5 1696290 G/R 11.12.8620.02.92 1 30.11.89 0 36 920891 G/R 213593 R/R 10.02.89 20.04.94 0 0 62313791 3+ R/R 28.02.87 21.02.92 0 0 60 878693 R/R 09.06.93 07.09.96 0 033 1107391 3+ R/R 01.08.87 29.02.92 0 0 55 1125690 R/R 20.08.86 13.10.930 0 86 120788 R/R 27.01.84 24.04.86 1 28.01.86 1 24.04.86 24 560992 3+R/R 13.04.92 04.04.93 1 10.04.92 1  4.4.93 0 1008692 2+ R/R 08.07.9211.07.94 1 17.08.93 1 11.07.94 13 1686490 3+ R/R 10.12.86 23.07.87 107.05.87 1 23.07.87 5 Patho Nr. Überleb Age stage Nod. Grade Men E-RecP-Rec 489292 59 71.7 1c 0 2 2 12 0 617792 27 1 639792 23 0 724493 4864.3 2 1 2 2 12 12 914593 33 86.2 2 2 2 8 9 935493 33 49.6 2 1 3 2 0 0963392 60 79.4 1b 0 2 2 3 12 979693 33 46.7 2 0 2 3 9 0 979893 48 77 4b1 2 2 1 0 1034792 56 43.5 3 1 2 1 0 6 1323293 48 61.4 2 1 3 2 0 01331693 36 71.2 2 1 2 2 6 1 1564593 56 65.3 2 1 2 2 0 0 78692 65 63.8 20 3 2 4 12 176989 84 43.4 2 1 2 1 273690 72 52.3 2 0 2 1 12 12 72528949.5 2 0 2 0 0 729991 57 65.7 2 1 2 2 12 12 733290 14 81.7 1b 0 2 2 1212 826790 91 77.7 2 0 2 2 9 0 867191 79 77.5 2 0 3 2 0 0 988590 90 61.51b 0 2 2 6 9 991790 68 50.7 1c 0 2 2 8 4 1031190 90 43.4 1c 0 2 11033391 55 62.4 2 1 3 2 12 12 1055592 79 77.8 2 0 2 2 6 12 1101191 7048.5 2 0 2 1 0 0 1426491 76 54.9 1b 0 3 2 0 0 1560492 42 50 1c 0 3 3 0 01605790 63 56.4 1c 1 2 2 12 6 1641488 109 67.4 1b 0 2 2 9 1121893 1659.9 2 1 3 2 4 2 1201592 29 48.9 1c 1 2 1 1 6 258093 102 79.4 1c 1 2 2479090 55 67.9 1c 0 2 2 0 0 806490 71 66.7 2 1 2 2 8 0 963589 60 47.8 21 3 1 0 972291 35 48.3 2 0 3 1 0 0 995589 48 58.4 3 1 3 2 6 1112389 1479.4 4b 2 2 2 0 0 1726892 40 38.7 2 0 3 1 0 0 289791 61 48.1 2 1 3 2 0 0337293 36 51.2 1c 0 2 1 6 2 879290 103 44.9 1c 0 3 1 1 6 893090 89 68.54 0 3 2 12 6 1106192 66 53.9 1c 0 2 2 2 12 1107789 77 50.3 1c 0 2 1 8 121113892 63 51.9 2 0 1 2 2 6 1118990 66 53.8 1c 0 2 2 6 4 1152692 59 57.72 1 2 2 4 12 1599789 19 80.2 1c 0 3 2 3 2 1614591 51 76.7 2 1 2 2 3 992390 73 45.8 2 1 3 1 0 1 99289 86 47.1 1c 1 2 1 130588 96 39 2 1 2 1306490 21 52.2 2 1 3 3 0 0 492191 84 56.2 2 1 3 2 3 0 529692 46 66.9 1b0 3 2 0 0 529990 17 77.1 2 1 3 2 0 0 538292 18 56.1 4 2 3 2 0 0 61409149 47.2 2 2 2 1 2 6 651591 76 49.3 3 1 2 1 3 9 673592 45 36.8 2 0 2 1 00 678092 49 56.7 1c 1 2 2 0 0 714289 71 55 1c 1 2 2 8 807492 54 1 84819316 58 2 2 2 2 0 4 955692 53 49.9 1c 1 3 3 2 1 1022090 32 51.7 2 2 2 2 121 1054987 23 71.5 2 0 3 2 1078192 39 79.4 2 1 3 2 1 9 1079689 93 81.4 42 3 2 1169792 47 67.3 2 1 3 2 6 9 1216692 48 45.3 2 1 2 1 4 6 1314689 7681.2 4 1 2 2 6 12 1391992 8 76 2 1 3 2 1696290 62 55.2 2 0 2 2 6 6920891 50.5 2 0 3 1 2 6 213593 62 45.3 1c 0 2 1 3 6 313791 60 68.6 2 1 32 0 0 878693 33 0 1107391 55 61.5 1c 0 2 2 3 1 1125690 86 43.1 3 1 3 1 00 120788 37 68.1 2 1 3 2 560992 12 59.1 4b 2 3 2 6 0 1008692 24 47.4 2 13 2 0 0 1686490 10 55.4 2 1 3 2 0 0

From FIG. 4, it is clear that the G388R mutation is to be found ingreater number in patients who already have metastases in the lymphnodes at the time of the first treatment. Of the patients with a G388Rmutation, 62.7% had lymph node metastases, while of the patients with noG388R mutation only 38.2% displayed metastases in the lymph nodes. Asthe lymph node metastasis status is an important prognostic marker forthe further discrimination of tumours with a worse and those with abetter prognosis, it can be concluded from this result that the G388Rmutation in the 85 patients studied leads to a more severe tumourprogression.

From FIG. 5 it is can be seen that in the group of patients studied, therelapse-free survival probability is very much lower for those with aG388R mutation than for those patients who have no G388R mutation. While74.4% of the patients with a relapse possess the 388R genotype, only25.6% have the 388G genotype. This shows that patients with the G388Rmutation suffer a relapse more quickly, and therefore could not besuccessfully treated.

In summary, it can be stated that the FGFR-4 mutation G388R leads to a2.7-fold (OR=2.7; CI: 1.02<OR<7.4) increased risk of metastasisformation in the lymph nodes and to a 5.44-fold (OR=5.44; CI:1.93<OR<7.4) increased risk of a tumour relapse. Patients with a mutatedFGFR-4 allele (G388R) thus seem to have a predisposition to a tumourrelapse and hence a poorer disease prognosis.

Materials Acrylamide Serva, Heidelberg Agar Difco, Detroit Agarose BRL,Eggenstein Ampicillin Boehringer, Mannheim Aprotinin Sigma, TaufkirchenN,N′-bisacrylamide Roth, Karlsruhe Caesium chloride BRL, EggensteinDesoxynucleotides Pharmacia, Freiburg Ethidium bromide Sigma,Taufkirchen Gelatine Sigma, Taufkirchen Guanidium isothiocyanate Fluka,Switzerland HEPES Serva, Heidelberg Sodium fluoride Sigma, TaufkirchenPMSF Sigma, Taufkirchen SDS Roth, Karlsruhe Tris Riedel de Haen, SeelzeTriton X100 Serva, Heidelberg Tween 20 Sigma, Taufkirchen

All substances not listed here came from the firms Sigma (Taufkirchen),Serva (Heidelberg), Riedel De Haen or Merck (Darmstadt) and the highestpossible purity grades were used.

Instruments Electrophoresis of DNA Workshop, MPI for Biochemistry,Martinsried Electrophoresis of Atto, Japan proteins Refrigeratedcentrifuge Biofuge 17S Heraeus, Hanau Protein transfer Semidry blotapparatus, Workshop, MPI for Biochemistry, Martinsried Sterile workbenchBiogard, The Baker Company, USA Cell culture Incubator B5060 EK/CO₂,Heraeus, Hanau Cell counting Coulter Counter, Coulter Electronics,Glasgow.

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Cell Line Origin No. U-138 ATTC HTB-16 U-373 ATTC HTB-17 U-172 U-118ATTC HTB-15 SF-763 SUGEN U-1240 SUGEN T-98G SUGEN U-937 ATCC CRL-1593SK-N-SH ATCC HTB-11 SH-SY5Y F. J. Klinz OAW-42 DKFZ PA-1 ATCC CRL-1572Caov-3 ATCC HTB-75 Hlac-78 Dr. Wustrow Hlac-79 Dr. Wustrow Scc-4 ATCCCRL-1624 Scc10a Dr. Wustrow Scc10b Dr. Wustrow Scc22a Dr. Wustrow Scc22bDr. Wustrow Scc17a Dr. Wustrow Sccl7b Dr. Wustrow FaDu Dr. Wustrow HaCatHBL100 ATCC HTB-124 MCF10A ATCC CRL-10317 SKBr-3 ATCC HTB-30 BT-549 ATCCHTB-122 MCF-7 ATCC HTB-22 BT483 ATCC HTB-121 T-47-D ATCC HTB-133 ZR-75-1ATCC CRL-1500 MDA-MB-468 ATCC HTB-132 MDA-MB-453 ATCC HTB-131 MDA-MB-361ATCC HTB-27 MDA-MB-415 ATCC HTB-128 MDA-MB-231 ATCC HTB-26 K-562 ATCCCCL-243

8 1 25 PRT Homo sapiens DOMAIN (1)..(25) amino acid sequence of FGFR-4(mutant) between positions 366-390 1 Arg Tyr Thr Asp Ile Ile Leu Tyr AlaSer Gly Ser Leu Ala Leu Ala 1 5 10 15 Val Leu Leu Leu Leu Ala Arg LeuTyr 20 25 2 25 PRT Homo sapiens DOMAIN (1)..(25) amino acid sequence ofFGFR-4 (wild-type) between positions 366-390 2 Arg Tyr Thr Asp Ile IleLeu Tyr Ala Ser Gly Ser Leu Ala Leu Ala 1 5 10 15 Val Leu Leu Leu LeuAla Gly Leu Tyr 20 25 3 29 DNA artificial sequence PCR primer for theamplification of FGFR-4 (wild-type and mutant) 3 gctcagaggg cgggcgggggtgccggccg 29 4 33 DNA artificial sequence PCR primer for theamplification of FGFR-4 (wild-type and mutant) 4 ccgctcgagt gcctgcacagccttgagcct tgc 33 5 24 DNA artificial sequence PCR primer for theamplification of the transmembrane domain of FGFR-4 (wild-type andmutant) 5 gaccgcagca gcgcccgagg ccag 24 6 23 DNA artificial sequence PCRprimer for the amplification of the transmembrane domain of FGFR-4(wild-type and mutant) 6 agagggaaga gggagagctt ctg 23 7 28 DNAartificial sequence primer for sequencing of the transmembrane domain ofFGFR-4 (wild-type and mutant) 7 gggaattcga ccgcagcagc gcccgagg 28 8 25DNA artificial sequence primer for sequencing of the transmembranedomain of FGFR-4 (wild-type and mutant) 8 gctctagaag agggaagagg gagag 25

What is claimed is:
 1. An isolated or purified mutated human fibroblastgrowth factor receptor-4 (FGFR-4), which comprises a point mutation inamino acid 388 of the wild-type human FGFR-4 having the amino acidsequence encoded by the nucleotide sequence of GenBank accession numberX57205, wherein the point mutation results in replacement of glycinewith arginine, and wherein the isolated and purified mutated humanFGFR-4 is characterized by overexpression in a human cell as compared tothe wild-type human FGFR-4 having the amino acid sequence encoded by thenucleotide sequence of GenBank accession number X57205 and/or tyrosinekinase activity in a human cell, which differs from the tyrosine kinaseactivity of the wild-type human FGFR-4 having the amino acid sequenceencoded by the nucleotide sequence of GenBank accession number X57205.